The long term objective of this proposal is to characterize in detail the structural and functional properties of zinc in biological systems. Zinc is the most common metal found in metalloproteins and is the only metal that is known to be required for every major class of enzyme catalysis. Hundreds of zinc proteins have been isolated and thousands of potential zinc binding sites have been identified in protein sequences. Imbalances in the levels of zinc or errors in its transport or regulation can have profound health consequence. Malfunctions in zinc homeostasis have been implicated in a wide range of diseases, including amyotrophic lateral sclerosis, Alzheimer's disease, and spongiform encephalopathies; as a modulator of synaptic transmission, zinc has been suggested to play a critical role in the proper functioning of the brain. Despite its importance, there is relatively little information available about biological zinc sites due to the difficulty of studying this spectroscopically "silent" metal. X-ray absorption spectroscopy, one of the few methods able to provide structural information for non-crystalline materials, will be used to determine the structures of zinc in a novel class of zinc-containing alkyl transfer enzymes and to characterize the relative importance of metal stereochemical preference and protein structure in defining the structure of a metal binding site. For the latter work, structures will be compared for a series of spectroscopically silent metal ions (Cu(l), Ag(l), Cd(ll), Hg(ll), Pb(ll), and As(lll), in addition to Zn(ll)). As a complement to "conventional" x-ray spectroscopy, new tools will be developed using high-resolution x-ray emission spectroscopy and zinc L-edge x-ray spectroscopy. In situ x-ray microprobe imaging and microspectroscopy will be used to characterize metal sites in intact biological tissue, and the newly developed tool of capillary electrophoresis/x-ray fluorescence will be used to determine the metal loading in metalloproteins, with particularly emphasis on the protein superoxide dismutase.